ObjectiveUmbilical cord milking (UCM) at birth may benefit preterm infants, but the physiological effects of UCM are unknown. We compared the physiological effects of two UCM strategies with immediate umbilical cord clamping (UCC) and physiological-based cord clamping (PBCC) in preterm lambs.MethodsAt 126 days’ gestational age, fetal lambs were exteriorised, intubated and instrumented to measure umbilical, pulmonary and cerebral blood flows and arterial pressures. Lambs received either (1) UCM without placental refill (UCMwoPR); (2) UCM with placental refill (UCMwPR); (3) PBCC, whereby ventilation commenced prior to UCC; or (4) immediate UCC. UCM involved eight milks along a 10 cm length of cord, followed by UCC.ResultsA net volume of blood was transferred into the lamb during UCMwPR (8.8 mL/kg, IQR 8–10, P=0.01) but not during UCMwoPR (0 mL/kg, IQR −2.8 to 1.7) or PBCC (1.1 mL/kg, IQR −1.3 to 4.3). UCM had no effect on pulmonary blood flow, but caused large fluctuations in mean carotid artery pressures (MBP) and blood flows (CABF). In UCMwoPR and UCMwPR lambs, MBP increased by 12%±1% and 8%±1% and CABF increased by 32%±2% and 15%±2%, respectively, with each milk. Cerebral oxygenation decreased the least in PBCC lambs (17%, IQR 13–26) compared with UCMwoPR (26%, IQR 23–25, P=0.03), UCMwPR (35%, IQR 27–44, P=0.02) and immediate UCC (34%, IQR 28–41, P=0.02) lambs.ConclusionsUCMwoPR failed to provide placental transfusion, and UCM strategies caused considerable haemodynamic disturbance. UCM does not provide the same physiological benefits of PBCC. Further review of UCM is warranted before adoption into routine clinical practice.

Therapeutic benefits of mesenchymal stem cells (MSCs) are now widely believed to come from their paracrine signalling, i.e. secreted factors such as cytokines, chemokines, and extracellular vesicles (EVs). Cell-free therapy using EVs is an active and emerging field in regenerative medicine. Typical 2D cultures on tissue culture plastic is far removed from the physiological environment of MSCs. The application of 3D cell culture allows MSCs to adapt to their cellular environment which, in turn, influences their paracrine signalling activity. In this study we evaluated the impact of 3D MSCs culture on EVs secretion, cargo proteome composition, and functional assessment in immunomodulatory, anti-inflammatory and anti-fibrotic properties.MSC-EVs from 2D and 3D cultures expressed classical EV markers CD81, CD63, and CD9 with particle diameter of <100 nm. There were distinct changes in immunomodulatory potencies where 3D cultures exhibited reduced indoleamine 2,3-dioxygenase (IDO) activity and significantly reduced macrophage phagocytosis. Administration of 2D and 3D EVs following double dose bleomycin challenge in aged mice showed a marked increase of bodyweight loss in 3D group throughout days 7–28. Histopathological observations of lung tissues in 3D group showed increased collagen deposition, myofibroblast differentiation and leukocytes infiltrations. Assessment of lung mechanics showed 3D group did not improve lung function and instead exhibited increased resistance and tissue damping. Proteome profiling of MSC-EV composition revealed molecular enrichment of EV markers (compared to parental cells) and differential proteome between EVs from 2D and 3D culture condition associated with immune-based and fibrosis/extracellular matrix/membrane organization associated function.This study provides insight into distinct variation in EV protein composition dependent on the cellular microenvironment of the parental cells, which could have implications in their therapeutic effect and potency. Overall, this work suggests that EVs produced from 3D MSC cultures did not enhance typical MSC-EV properties expected from 2D cultures (immunomodulation, anti-fibrotic, anti-inflammatory). The outcome highlights critical differences between MSC-EVs obtained from different culture microenvironments, which should be considered when scaling up MSC culture for clinical manufacturing.

Objective— The objective of this study was to investigate the effect of intravenous maternal sildenafil citrate (SC) administration on vascular function in growth-restricted fetal sheep. Approach and Results— Fetal growth restriction (FGR) results in cardiovascular adaptations that redistribute cardiac output to optimize suboptimal intrauterine conditions. These adaptations result in structural and functional cardiovascular changes, which may underlie postnatal neurological and cardiovascular sequelae. Evidence suggests SC, a potent vasodilator, may improve FGR. In contrast, recent clinical evidence suggests potential for adverse fetal consequence. Currently, there is limited data on SC effects in the developing fetus. We hypothesized that SC in utero would improve vascular development and function in an ovine model of FGR. Preterm lambs (0.6 gestation) underwent sterile surgery for single umbilical artery ligation or sham (control, appropriately grown) surgery to replicate FGR. Ewes received continuous intravenous SC (36 mg/24 h) or saline from surgery until 0.83 gestation. Fetuses were delivered and immediately euthanized for collection of femoral and middle cerebral artery vessels. Vessel function was assessed via in vitro wire myography. SC exacerbated growth restriction in growth-restricted fetuses and resulted in endothelial dysfunction in the cerebral and femoral vasculature, irrespective of growth status. Dysfunction in the cerebral circulation is endothelial, whereas smooth muscle in the periphery is the origin of the deficit. Conclusions— SC crosses the placenta and alters key fetal vascular development. Extensive studies are required to investigate the effects of SC on fetal development to address safety before additional use of SC as a treatment.

Positive end expiratory pressure (PEEP) improves oxygenation in mechanically ventilated preterm neonates by preventing lung collapse. However, high PEEP may alter cerebral blood flow secondary to the increased intrathoracic pressure, predisposing to brain injury. The precise effects of high PEEP on cerebral haemodynamics in the preterm brain is unknown. We aimed to assess the effect of PEEP on microvessels in the preterm brain using synchrotron radiation (SR) microangiography which, enables in vivo real-time high-resolution imaging of the cerebral vasculature. Preterm lambs (0.8 gestation, n=4) were delivered via caesarean section, anaesthetised and ventilated. SR microangiography of the right cerebral hemisphere was performed with iodine contrast administered into the right carotid artery, during PEEP ventilation of 5 and 10 cmH2O respectively. Carotid blood flow was measured using an ultrasonic flow probe placed around the left carotid artery. An increase of PEEP from 5 to 10 cmH2O increased the diameter of small cerebral vessels (<150 µm), but decreased diameter of larger cerebral vessels (>500 µm) in all 4 lambs. Additionally, the higher PEEP increased the cerebral contrast transit time in 3 of the 4 lambs. Carotid blood flow increased in 2 lambs which, also had increased carbon dioxide levels during PEEP 10. Our results suggest that PEEP of 10 cmH2O alters the preterm cerebral haemodynamics, with prolonged cerebral blood flow transit and engorgement of small cerebral microvessels likely due to the increased intrathoracic pressure. These microvascular changes are generally not reflected in global assessment of cerebral blood flow or oxygenation.

No abstract

Erythropoietin (EPO) is being trialled in preterm infants to reduce brain injury, but high doses increase lung injury in ventilated preterm lambs. We aimed to determine whether early administration of lower doses of EPO could reduce ventilation-induced lung injury and systemic inflammation in preterm lambs. Ventilation was initiated in anaesthetized preterm lambs [125 ± 1 (SD) days gestation] using an injurious strategy for the first 15 min. Lambs were subsequently ventilated with a protective strategy for a total of 2 h. Lambs were randomized to receive either intravenous saline (Vent; n = 7) or intravenous 300 ( n = 5), 1,000 (EPO1000; n = 5), or 3,000 (EPO3000; n = 5) IU/kg of human recombinant EPO via an umbilical vein. Lung tissue was collected for molecular and histological assessment of inflammation and injury and compared with unventilated control lambs (UVC; n = 8). All ventilated groups had similar blood gas and ventilation parameters, but EPO1000 lambs had a lower fraction of inspired oxygen requirement and lower alveolar–arterial difference in oxygen. Vent and EPO lambs had increased lung interleukin (IL)-1β, IL-6, and IL-8 mRNA, early lung injury genes connective tissue growth factor, early growth response protein 1, and cysteine-rich 61, and liver serum amyloid A3 mRNA compared with UVCs; no difference was observed between Vent and EPO groups. Histological lung injury was increased in Vent and EPO groups compared with UVCs, but EPO3000 lambs had increased lung injury scores compared with VENT only. Early low-doses of EPO do not exacerbate ventilation-induced lung inflammation and injury and do not provide any short-term respiratory benefit. High doses (≥3,000 IU/kg) likely exacerbate lung inflammation and injury in ventilated preterm lambs. NEW & NOTEWORTHY Trials are ongoing to assess the efficacy of erythropoietin (EPO) to provide neuroprotection for preterm infants. However, high doses of EPO increase ventilation-induced lung injury (VILI) in preterm lambs. We investigated whether early lower doses of EPO may reduce VILI. We found that lower doses did not reduce, but did not increase, VILI, while high doses (≥3,000 IU/kg) increase VILI. Therefore, lower doses of EPO should be used in preterm infants, particularly those receiving respiratory support.

Fetal growth restriction induces a haemodynamic response that aims to maintain blood flow to vital organs such as the brain, in the face of chronic hypoxaemia r Maternal sildenafil treatment impairs the hypoxaemia-driven haemodynamic response and potentially compromises fetal development.

No abstract